IV.

How Scientists Work with Genes

Scientists have developed a number of biochemical and genetic techniques by which DNA can be separated, rearranged, and transferred from one cell to another. Some of these laboratory methods help scientists study the properties of genes in nature—for example, by comparing DNA from different animals to find out whether those animals are closely related to each other or only distant relatives. Other DNA techniques provide tools for genetic engineering—the alteration of genes in an organism. These tools are used in industry to develop commercial products, such as hardier crops, microbes that can break down oil slicks or decompose garbage, and improved medicines.

A.

Recombinant DNA

The DNA molecules of all life forms, from oak trees to sea horses, have the same structure and the same four bases. Scientists have made use of these similarities in a technology called recombinant DNA. In this laboratory method, one or more genes of an organism are introduced into a second organism. The new genes, sometimes known as foreign DNA, become functional in the second organism and produce a desired protein. In this way, scientists can create changes in the genetic makeup of an organism that would be unlikely to occur through natural processes.

Scientists use recombinant DNA when they want to obtain large amounts of a protein, such as insulin, produced by a gene. Insulin was once in short supply for diabetics, whose bodies lack adequate supplies. Insulin supplies were derived from cows in an expensive and time-consuming process. Today recombinant DNA techniques produce insulin cheaply and in abundance. The first step in creating insulin using recombinant DNA is to isolate the sequence of nucleotides in the DNA of a human cell that forms the insulin gene. Scientists use restriction enzymes, specialized proteins that act like molecular scissors, to cut the double-stranded DNA at the point where the insulin gene occurs. The isolated DNA can then be recombined, or spliced, with a vector, a fragment of DNA that is able to transport genes from one organism to another. A vector may be a plasmid, a small, circular segment of DNA found in bacteria. Bacteriophages, viruses that are parasites of bacteria, also act as vectors.

Scientists insert the vector containing the insulin gene into a bacterium, such as E. coli. Within just a few hours, a single E. coli will reproduce hundreds of times to make millions of cells, all containing exact copies of the insulin-producing gene inserted by the scientists. This process of making many cells with identical DNA is known as cloning.

B.

DNA Libraries

A DNA library is a storehouse of genetic information maintained in bacteria instead of books. These bacteria are clones created by recombinant DNA, and the foreign DNA they hold is the library’s store of information. DNA libraries are helpful to scientists who require a plentiful supply of particular DNA segments to do their work. These repositories of genetic information are stored in small tubes, which can easily be shipped to other researchers for study.

Each library has a unifying theme. For example, a library may contain the entire chromosomal DNA, or genome, of a given organism, or it may consist of genes that are active within certain types of cells, such as heart cells. To create a library of the human genome, DNA from all the human chromosomes would be cut into many pieces. These pieces would be randomly inserted into vectors, such as plasmids, which would then be placed into a population of bacteria. Taken together, the entire population of bacteria would contain all the DNA of the human chromosomes.

C.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) offers an alternative to vector-based cloning as a means of generating numerous copies of DNA from a small initial sample. Performed in a test tube, PCR mirrors the way in which DNA is replicated within a cell. To perform PCR, scientists isolate the piece of DNA to be amplified (multiplied) in a test tube and heat it to separate the two strands of the molecule. As cooling occurs, short pieces of DNA called primers are added to the test tube. The primers attach to each strand, marking the segment that will be cloned. Free-floating nucleotides and an enzyme called DNA polymerase are then added to the mixture. DNA polymerase uses the free-floating nucleotides to build a complementary copy of each amplified DNA segment, resulting in two new double-stranded DNA molecules. Each cycle of heating and cooling doubles the amount of the desired DNA fragment in the test tube. In a matter of hours, scientists can obtain millions of copies of a desired piece of DNA. PCR enables scientists to amplify traces of DNA found at a crime scene or in a fossil animal to produce sufficient quantities to study.

D.

Gel Electrophoresis

PCR and recombinant DNA techniques create large amounts of DNA segments. To study the structure of these segments, researchers use a process known as gel electrophoresis. This technique can be used to identify genes in humans that have previously been identified in other organisms, such as fruit flies. It can also be used to compare the DNA found from blood or hair samples at a crime scene with the DNA of a suspect in the crime. In gel electrophoresis, restriction enzymes break up the DNA under study into restriction fragments of varying lengths. Solutions containing these fragments are placed within a thick gel. An electric current is applied to the gel, causing one end of the gel to have a positive charge and the other to have a negative charge. All of the restriction fragments begin to move from the negative end of the gel toward the positive end. The smaller fragments move faster than the larger fragments. When the current shuts off, typically after several hours, the DNA fragments have spread out across the gel, with the smaller ones closer to the positive end. The dispersed fragments display a pattern resembling a bar code. Each bar in this pattern contains DNA fragments of a certain size. Scientists can identify specific restriction fragments by their location on the gel. A complementary sequence of DNA can be used as a probe to find a restriction fragment on the gel that has a particular nucleotide sequence. Scientists may use DNA found in blood at a crime scene as the probe to see if it pairs up with any of the DNA fragments in the gel electrophoresis. If pairing occurs, the DNA from the crime scene is from the same person who provided the DNA sample for the gel electrophoresis.

E.

DNA Sequencing

Once an interesting piece of DNA has been isolated or identified, scientists often need to determine if the sequence of nucleotides in the fragment is related to known genes and to determine what kind of protein it might make. Scientists use DNA sequencing to detect genetic mutations linked to diseases such as cystic fibrosis. Scientists have also used this method to alter the sequence of a gene and study the function of the resulting protein. In DNA sequencing, scientists create many copies of a single-stranded DNA fragment that will be used to synthesize a new DNA strand. An equal number of copies of the fragment are placed into four different test tubes to act as the template for the synthesis of a new strand. The enzyme DNA polymerase and free nucleotides are added to each test tube. Each test tube also receives one type of dideoxy nucleotide—a nucleotide that closely resembles either adenine, guanine, thymine, or cytosine. These nucleotides can attach to the end of the new complementary DNA strand, but they cannot bind to anything else, thus they terminate the synthesis of the new DNA strand.

DNA polymerase uses the free nucleotides to build a complementary DNA strand. If the original DNA fragment contains guanine, DNA polymerase delivers a cytosine dideoxy nucleotide to pair with the guanine base on the original strand. The cytosine links with the growing chain of nucleotides on the complementary DNA strand, but it is unable to bind with any other nucleotide. The newly formed DNA fragment terminates with the cytosine dideoxy nucleotide at the end of the chain. The reactions in each of the four test tubes produce a series of DNA fragments in which the new strands terminate at a known base. Each test tube produces fragments that differ in length from the other test tubes. The newly formed fragments are sorted in an electrophoresis gel that can detect differences as small as one nucleotide in length. By analyzing these sorted fragments, scientists can determine the complementary base sequence for the original DNA fragment. This sequencing method has become a routine laboratory technique, automated with specialized machines and computers that can prepare DNA samples and read nucleotide sequences far faster and more accurately than people can.

F.

Gene Chip

The gene chip, also known as a DNA chip or DNA microarray, is a thumbnail-sized chip of glass or silicon that carries DNA instead of electronic circuits. Gene chips can identify the genes that are active within a cell and help identify mutated genes. In one application, scientists take a single strand of DNA that contains a defective gene and use ultraviolet light to attach the strand onto a glass or silicon chip. A second DNA strand isolated from a patient is attached to fluorescent markers and deposited onto the chip. If the patient’s DNA strand bonds with the DNA already bonded to the chip, then the individual’s DNA contains the defective gene. When the DNA on the chip pairs with the fluorescent DNA, it develops a fluorescent glow that can be viewed with a microscope and interpreted by a computer. A diagnostic gene chip may soon be manufactured to hold the DNA sequences of all the known disease-causing genes, making diagnosis for genetic disorders fast, reliable, and inexpensive. Gene chips also distinguish between active DNA—DNA that is being transcribed to produce mRNA—and inactive DNA. Researchers use these chips to learn how the transcription of a group of genes is affected when cells are exposed to a drug.